Enzymatic reactors are finding employ in leukemia therapy, dialysis, and hepatic support. In addition, whole microbial cells are being tested and systems developed for their suitability as artificial organs.
An artificial organ is a device that will hopefully replace or augment the diseased, damaged, or malfunctioning one. By and large, these devices are only able to perform one or two of the vital functions of the replaced or assisted human organ. One of the most studied artificial organs--as well as the most widely used--is the artificial kidney (or dialyzer). In this country alone, about 60,000 people die annually of renal failure. The only effective means of treating kidney ailments today is through the use of dialysis. Unfortunately, artificial kidneys are not as efficient nor as convenient as they should be.
To maintain the consistency of the human internal environment, the actual kidney performs several functions: It detoxifies certain organic compounds; it synthesizes both hormones and enzymes; it excretes waste; and it also maintains the balances for water, electrolytes, and acids and bases. In performing these body functions, the kidney regulates the concentration of most of the plasma constituents; these include urea, uric acid, creatinine, phenols, water, and the ions of sodium, potassium, calcium, magnesium, bicarbonate, chloride, phosphate, and sulphate.
Some 32,000 patients are treated by dialysis in the U.S. The artificial kidney supplements and in most cases performs extensively, one of the major body functions--the removal of certain potentially toxic substances all of which are soluble in water, from the body. At times, the dialyzer is also called upon to remove excess body water; this is typically removed by ultrafiltration techniques (the imposition of negative pressure on the blood within the dialyzer). However, since we do not know what the exact toxic substances are, we cannot determine if they are adequately removed. (Three of the currently accepted uremic toxin markers are urea, creatinine, and uric acid). Moreover, these artificial kidneys are non-physiologic and use passive mass transfer, instead of the complex, nephronic processes. This limits the efficiency of the therapy, so the search for more physiologic and efficient processes is still ongoing.
The supply system for the dialyzer typically is a one-pass type. A saline concentrate is mixed with deionized water to produce a set flowrate of influent dialysate. The supply is preheated to 37 .degree.-39 .degree. C. to maintain the blood temperature in the extracorporeal circuit. The produced dialysate is continuously monitored to insure that it is at the correct concentration. A batch system (which requires a large tank of premixed solution), on the otherhand, requires an initial supply of 120-400 liters of deionized water. Such a large supply volume might be an inconvenience for the hospital to provide, but it is virtually impossible for the home dialysis patient to maintain.
For several years now, adsorption devices have been available to provide a dialysate supply system that utilizes a rather small volume (5-30 liters) of water, compared to 120-400 l volumes required for the standard dialysis procedure. The sorbents employed include activated carbon, zirconium phosphate, zirconium oxide, alumina, along with urease. By and large, these systems are limited in their ability to remove urea from the dialysate solution, even with the urease enzymes present. The increased urea concentration therefore reduces the driving force available to effect mass transfer from the blood. Examples of such systems are suggested by Gordon, Bergstrom, Rosenfeld & Maxwell in Adsorption of Uremic Toxins. VI International Congress of Nephrology; Florence, Italy, June, 1975, and Maeda, Ohta, Manji, Saito, Kawaguchi, Amano, Shibata, & Kobayashi. Dialysate regeneration: 30 liter dialysate supply system with sorbents. Kidney International. 10, S 289-S 295 (1976 ).
Around 1966, CCI Life Systems, Inc. employed a system which uses about 5.5 liters of dialysate. It is a self-contained unit (once the initial water is supplied) that maintains the dialysate concentration within normal limits. The metabolic wastes products contained by the returned dialysate are absorbed by the cartridge so that no build-up of uremic toxins occurs. The regeneration system is comprised of activated carbon, zirconium phosphate, zirconium oxide, and urease. The urease converts urea to ammonia, which together with calcium and magnesium ions, is exchanged for sodium and hydrogen ions on the zirconium phosphate. Phosphate is removed via the zirconium oxide, and the activated charcoal removes other organic metabolites.
There are some major problems with this system. First of all, the dialysate flow cannot exceed approximately 250 ml/min due to the excessive pressure drop through the cartridge. This requires a lengthened dialysis treatment; an increase in time the patient must be connected to the dialyzer. In addition to this problem, the removal of metabolic waste products from the dialysate are not complete, and ammonia builds up, further decreasing the rate of dialysis.
Paul Malchesky and Yokihiko Nose of the Cleveland Clinic Foundation have been examining the use of microbial reactors as artificial kidneys. Malchesky and Nose hope to culture a bacterial population that can remove and possibly recycle urinary waste products. Since the solute removal is a function of the medium composition, the removal rates could be adapted to the patient's requirements.
Some literature concerning this development by Malchesky and Nose includes Malchesky PS & Nose Y. 1977. Biological Reactors for Renal Support. Presented at the 23rd Annual Meeting of the Amer. Soc. Art. Inter. Organs. Montreal, Canada. 22 April; Malchesky PS and Nose Y. 1975. Biological Reactors as Artificial Organs. Cleveland Clinic Quarterly. 42,3:267-271; Malchesky PS and Nose Y. 1974. The Use of Biological Reactors as Artificial Organs. Presented at the 27th Ann. Conf. Eng. Med. Bio. Philadelphia, Pa. October 6-10; and Malchesky PS, Fingerhood B., Nose Y., Gavan T., & Willis C. 1976. The Use of Microorganisms for Renal Support. Presented at the 29th Ann. Conf. Eng. Med. Bio. Boston, Mass. November 6-10.
Their method involved the culturing of activated sludge bacteria and some supplementary species (i.e. Rhizobium) with normal urine. While the ideal support system might be subjected to different quantities of metabolic by-products (since normal, as opposed to uremic urine is used), the actual requirements should be identical.
The basic system uses a stirred aerobic vessel containing 900 ml of urine and an inoculum of 5.times.10.sup.5 bacteria. Temperatures ranged from 20.degree.-37 .degree. C. Each batch culture was maintained for 3-4 daily pH adjustments. This culture then served as the inoculum for the subsequent reactor.
To date, some 110 systems have been studied. They found that urea and uric removals were substantial (74 and 64%, respectively) and the creatinine reduction was 25%. Almost half the time, the urea was completely removed from the culture. Malchesky and Nose attribute the failure to consistently remove urea completely to contamination or a build-up of ammonia between pH adjustments. (For those times that urea was completely removed, higher uric acid and creatinine removals were obtained).
Overall then, Malchesky and Nose found that their cultures removed 6.6 g/d of urea, 163.5 mg/d of creatinine, and 145.5 mg/d of uric acid. Excess calcium, phosphate, and potassium were also removed, but to a smaller degree. In addition, they found that there were adptation periods required before the bacterial density increased. These studies were designed to verify culture selection and not the final consumption rates. Now that they have adequate bacterial stock cultures, they will begin continuous culture studies to determine the system feasibility.
While the utilization of one biological system (bacteria) that requires the waste products of another living thing (human being) is a symbiotic, physiologic relationship, the potential problems with such a system are numerous. The most important of these are the possible infections that could result from the utilized bacterial system which can come into contact with the patients blood supply or from possible pyrogenic reactions within the patient.
Asher and his co-workers at Exxon have been developing an adsorbent system to remove toxins from the gastro-intestinal tract. For instance, see Asher W. J., Vogler T. C., Bovee K. C., Holtzapple P. G., & Hamilton R. W. 1976. Projections and measurements of in vivo performance of liquid membrane capsules. Kidney International. 10:S2540258; Asher W. J., Vogler T. C., Bovee K. C., Holtzapple P. G. & Hamilton R. W. 1976. In vivo performance of liquid membrane capsules. Trans. Amer. Soc. Artif. Intern. Organs. XXII; and Asher W. J., Vogler T. C., Bovee K. C., Holtzapple P. G., & Hamilton R. W. 1977. J. Dial. 1, 3:261-284.
They have been working with liquid membrane capsules (LMC). These LMC contain stabilized drops of emulsion suspended in a continuous phase. The capsule diameter ranges about 375 microns, with microdroplets of 1 to 5 micron diameter in their interior.
Urea diffuses from the blood into the intestine. The urease enzyme in the capsule converts the transported urea to carbon dioxide and ammonia. The ammonia is trapped within the LMC and excreted, while the carbon dioxide is eliminated by the lungs.
To obtain effective trapping of the uremic "toxin", the following criteria must be met:
1. Mucosal membrane transport must be sufficient. PA0 2. The LMC should not greatly affect the mucosal membrane transport. PA0 3. The LMC must remove the toxin under the conditions present within the intestinal lumen. PA0 4. The LMC should not damage the intestinal mucosa. PA0 5. The urease enzyme must be protected from the acidic stomach environment. PA0 6. The LMC should be stable to bile concentrations. PA0 7. Pancreatic secretions, due to their proteolytic activity, should not reduce the urease activity below the level required.
The first four requirements have been met by the general nature of the LMC. To prevent any damage to the urease in the stomach and to maintain the LMC effectiveness in the intestine, the oil phase contains monoolein and two different types of LMC are used. One LMC encapsulates urea and the other encapsulates the citric acid, which is the ammonia trapping agent.
The pancreatic secretion releases the urease enzyme from the LMC, where it is exposed to the secretion's proteolytic activity. These effects seem to be counter--balanced so far--a higher concentration of pancreatic secretion releases more urease, which is then exposed to a higher proteolytic activity. Therefore, a constant urease concentration is available for urea converstion.
The expected urea removal is on the order of 24 g/d for humans. (To date, the LMC formulations have only been tested on dogs). Thus, a milk shake comprised of these LMC might prove to be another valuable adjunct to dialysis. Additional LMC might be developed to handle other uremic toxins.
TMC Chang at McGill has been studying "artificial cells" for well over 2 decades. The cells are in the form of micro encapsulated enzymes, cells, cell extracts, adsorbents or other biologically active materials. The encapsulating membranes have a molecular weight cutoff of about 5000, a thickness of 200 A, and a large surface area to volume ratio.
Chang has used these cells to remove urea from the body (microencapsulated urease); treat mice with acatalasemia, a congenital enzyme deficiency (microencapsulated catalase); treat lymphosarcoma (microencapsulated asparaginase); and remove toxins and waste products (microencapsulated absorbents).
For instance, see Chang TMS. 1974. A Comparison of Semipermable Microcapsules and Standard Dialyzers for Use in Separation. Sep. Purif. Meth. 3, 2: 245-262; Chang TMS. 1975. Microencapsulated adsorbent hemoperfusion for uremia, intoxication, and hepatic failure. Kidney International. 7; S387-S392; Chang TMS. 1976. Microcapsule artificial kidney. Including updated preparative procedures and properties. Kidney International. 10:S218-S224; and Chang TMS. 1976. Hemoperfusion alone and in series with ultrafiltration or dialysis for uremia, poisoning, and liver failure. Kidney International. 10:S305-S311.
Chang has recently combined the first and last techniques to improve on the dialysis procedure.
Chang has found that the clearances for a renal support device are 230 ml/min for creatinine, 100 ml/min for middle molecules, and greater than 200 ml/min for toxic drugs. These values are from two to ten times greater than the rates achieved with conventional treatment. The microencapsulated urease is capable of reducing the blood urea concentration by 50% within 90 minutes. The urea is converted to ammonia with the microencapsule, so an ammonia trapping agent is required, as Asher's method does.
Because the ammonia trapping agent was a fairly new development, Chang has only reported clinical data for the use of the microencapsulated absorbent (activated charcoal system) in conjunction with a 0.2 m.sup.2 (membrane area) ultrafilter. He found that 2 hours of hemoperfusion with this system was as effective as 6 to 8 hours of treatment with a standard (1-2 m.sup.2) dialyzer.
The system suggested by Chang is somewhat similar to the system by CCI Life Systems referrd to hereinabove. There are many other experimental programs using enzymes and bacteria in dialysis. For example, Ioakim and Rosen at the University of London are working on a urease--ion exchange--carbon system.
For a discussion of the use of enzymes, see the article by applicant entitled Microbial and Enzymatic Systems Serve as Artificial Organs, Ackerman, R.A. SIM News, Sept., 1977, p. 4 ff.
An object of the present invention is to provide a microbial system for converting urea and especially a microbial system as a dialysate regenerator, which system is capable of digesting the waste products of the patient. A further object of the present invention is to provide a dialysate supply system capable of providing a sterile dialysate at normal flow rates (e.g., about 500 ml/min) to any currently available dialyzer.
The present invention makes it possible to employ a low-volume dialsate supply device (e.g., about 10 leters of water or less) while providing for relatively high dialysate flow rates (250--1000 ml/min) as compared to currently available low-volume dialysate supply devices.
By employing the present invention, a sterile dialysate is dispensed. According to the present invention, certain nitrogen-metabolizing strains of bacteria are employed which degrade the chemicals in the dialysate that were removed from the blood. The bacteria employed is capable of degrading nitrogenous compounds to gaseous products, leaving no residue within the culture.